Attenuated cyclooxygenase-mediated vasodilation in cutaneous microvasculature with no difference in platelet aggregation in women with endometriosis

The institutional Review Board at The Pennsylvania State University approved all experimental procedures and protocols. Verbal and written informed consent were voluntarily obtained from all participants before participation and in accordance with the guidelines set forth by the Declaration of Helsinki ( NCT05962034 ). These data were collected in the same experiment protocol as detailed in Williams & Alexander, 2025 ( Williams & Alexander, 2025 ). The protocols described herein occurred following the previously published protocol. All potential participants underwent screening to include a health history questionnaire, physical examination, medical screening, and a blood chemistry analysis (Chem 24, Quest Diagnostics, Pittsburgh, PA). Twelve women with clinically diagnosed endometriosis (nine diagnosed with laparoscopy, three diagnosed by a gynecologist from symptomology, ultrasound imaging, and family history) and nine healthy women participated in the study. Participants were free of cardiovascular incident history and lacked neurological and dermatological disease. The majority of participants were not using over-the-counter or prescription medications with known primary or secondary cardiovascular or hematologic effects such as hormone replacement therapy, statins, or anticoagulants at the time of testing; one participant with endometriosis was utilizing spironolactone but had no history of hypertension or edema. At the time of participation, participants had not used non-steroidal anti-inflammatory drugs in ≥ 2 weeks or norepinephrine-dopamine reuptake inhibitors within 24 hours. Participants did not use tobacco products. All participants were born with a uterus and at the time of testing had at least one intact ovary. Data reported here was collected in tandem with those reported in our recent publication, Williams & Alexander (2025) . Each participant underwent two total experimental visits that included the protocols reported in that publication as well as the protocols reported here. Following those sympathoexcitatory maneuvers, recovery to baseline blood pressure and heart rate were identified before moving on to the anodal stimulation protocol (described here), and a stable cutaneous blood flow was measured for 10 min before moving on to the stimulation protocol. Participants were instructed to avoid caffeine, alcohol, fish oil, and garlic for 24 hours prior to their experiment visits. We utilized a randomized, placebo-controlled, single-blind experimental design. Experiments took place at least 2 weeks apart. Participants were given a capsule of either 650 mg aspirin, a non-selective cyclooxygenase inhibitor, or placebo and were not told which they had taken until completion of participation. Anodal stimulation procedures took place ~2.25 hours after participants ingested the capsule ( Nagelschmitz et al., 2014 ). Blood samples for plasma testing and platelet aggregometry were taken immediately after the anodal stimulation procedures. Given the variation of menstrual cycles in the endometriosis population, the use of IUDs in both groups, and the hysterectomies reported in the endometriosis group, participants were tested without regard to menstrual cycle. Participants were seated upright and resting with their right arm extended, palm facing up, on a table at approximately heart-level. An iontophoresis chamber (MIC-ION1R-P1, Moor Instruments, Wilmington, DE) was placed on the ventral aspect of the forearm distal to the antecubital fossa. We filled the chamber with lactated Ringer’s solution and placed a laser Doppler flowmetry probe (VP12; Moor Instruments, Wilmington, DE) in the center of the chamber to continuously monitor red blood cell (RBC) flux (perfusion units, PU). A conductive pad (MIC-CP-V2, Moor Instruments) was placed on the ventral aspect of the ipsilateral wrist ~10 – 15 cm apart depending on the length of the forearm. The chamber and conductive pad were connected to an iontophoresis controller (VMS-ION, Moor Instruments). Baseline RBC flux was continuously recorded for 10 minutes. At the end of the baseline phase, a 50 μAmp current was administered to the skin followed by a rest phase with no current application. The following stimuli increased by increments of 50 μAmp in a step-wise, “on-off” manner up to 200 μAmp with each stimulation lasting 2 min and each rest phase lasting 3 min. Following the final stimulus and rest phase, we monitored local skin blood flow for a 15-minute recovery period with no current being applied. Blood pressure was taken on the contralateral arm (Connex Spot Monitor, WelchAllyn, Skaneateles Falls, NY) at the end of the baseline and rest phases and every 5 minutes during the recovery phase. Cutaneous vascular conductance was calculated as the quotient of RBC flux and mean arterial pressure (calculated from brachial blood pressure measurement). Baseline RBC flux was averaged over the entire stable 10-min period, an average of the final 30 seconds of each stimulus or rest phase, and the 30 sec leading to the end of each 5 min period of the recovery phase was used for point-to-point analysis ( See Figure 1 , X axis ). A research nurse collected a venous blood sample at the antecubital region in a K 2 EDTA-coated tubes for plasma isolation which was centrifuged at 4,000 RPM for 15 min at room temperature (Quest Diagnostic Horizon Centrifuge, Pittsburgh, PA) and aliquoted into 1.0 ml cryovial to be stored at −80 °C for later analysis. The concentration of COX metabolites Thromboxane B 2 (TXB2) and Prostaglandin 2 (PGE2) were measured in the serum samples using TXB2 ELISA Kit (Cat. No. ADI-900-002; Enzo Life Sciences) and PGE2 ELISA Kit (Cat. No. ADI-900-001; Enzo Life Sciences), respectively. Measurements were obtained according to the manufacturer’s guidance, and the concentration of TXB2 and PGE2 were calculated as follows: 1) Calculated the average net Optical Density (OD) bound for each standard and sample by subtracting the average non-specific binding (NSB) OD from the average OD bound. 2) calculated the binding of each pair of standard wells as a percentage of the maximum binding wells (B0) using the following formula: Percent Bound = (Net OD/ Net B0 OD) x 100. 3) Plot Percent Bound versus Concentration of TXB2/PGE2 for the standards. We calculated and plotted the standard curve accordingly. The concentration of TXB2/PGE2 in the unknows was determined by interpolation. Following collection of blood for plasma isolation, peripheral whole blood samples (~ 5.4 ml) were collected sodium citrate-lined blood sample tubes (9:1 ratio, BD Vacutainer ® , Franklin Lakes,NJ, USA) and analyzed within 3 hours following blood draw. Samples were handled carefully to avoid jostling. Blood samples were analyzed using whole blood impedance aggregometry (CHRONO-LOG ® Model 700 WholeBlood/Optical Lumi-Aggregometer and associated AGGRO/LINK ® 8 software; Chrono-log Corporation, Havertown, PA). Procedures for this technique are detailed elsewhere ( Williams et al., 2024 ). Briefly, 500 μl of the blood sample was incubated at 37 °C in 500 μl isotonic irrigation grade saline or 500 μl Terutroban solution (2.67x10 −7 M; Sigma–Aldrich, Darmstadt, Germany, SML1198) to block thromboxane receptors in a 1 ml cuvette. A stir bar was also placed and the cuvette was moved to a magnetic stirrer well within the aggregometer (1200 RPM). A two-pronged electrode was placed in the sample, and the electrical resistance between the two probes was continuously recorded with baseline resistance set to 0 Ω. A coagulation agonist was added to the sample to induce platelet aggregation around the prongs and increase resistance. We utilized four agonists: 1 μl of collagen at 1 mg/ml (col 1), 5 μl of collagen at 1 mg/ml (co 5l), 10 μl arachidonic acid at 50 mM (AA; Chrono-log, product numbers 385 Collagen and 390 AA), and 10 μl U46619 28.5 mM (Tx10; MedChemExpress, NJ, USA). Following addition of the agonist, resistance was recorded for 6 minutes, and the total aggregation (area under the curve; AUC; trapezoid rule) was continuously calculated and used for analysis. Each agonist – col 1, col 5, AA, and Tx10 – were tested in both the saline and Terutroban solutions. RBC flux was continuously recorded at 1000 Hz and stored for offline analysis (PowerLab and LabChart, ADInstruments, Bella Vista, NSW, Australia). CVC data were analyzed with linear mixed effects model corrected for multiple comparisons with Tukey’s method (SAS v. 9.4; Cary NC). To investigate any related factors leading to COX-mediated vasodilation, AUC was calculated for CVC response to anodal stimulation, and simple linear regression analyses were conducted relating platelet count, TxB2 concentration, and PGE2 concentration to AUC (Prism v8.1, GraphPad Software, La Jolla, CA). Platelet aggregation characteristics were analyzed by three-way ANOVA (SAS v. 9.4). If aggregation did not occur in response to a reagent, Lag was forced to 361 seconds (1 second beyond the recording period). To account for a possible effect of platelet count ( Femia et al., 2013 ), platelet aggregation characteristics were normalized to platelet count (i.e., calculating the quotient of the outcome characteristic and the platelet count) and re-analyzed as stated previously. Plasma samples were analyzed with two-way ANOVA. Plasma volume was estimated using the Hakim formula for females [ p l a s m a v o l u m e = ( 1 − H c t ) ∗ ( 1530 + ( 47.9 ∗ b o d y w e i g h t ) ) ] ( Ismail et al., 2001 ) and compared between groups with an unpaired t-test. TXB2 and PGE2 concentrations were assessed for differences between groups and treatments with two-way mixed-effects ANOVA (SAS v. 9.4). Outliers were determined via the ROUT method (Q = 1%) for each outcome. For all analyses, significance was set at p ≤ 0.05. Once significant differences were identified, Cohen’s d or Cohen’s f effect sizes were calculated as appropriate.

Participant characteristics are shown in Table 1 . Participants were generally well matched. Platelet counts, while remaining within clinically acceptable ranges, were significantly higher in women with endometriosis compared with HC (p = 0.04, d = 0.98). Results of anodal stimulation are shown in Figure 1 . Women with endometriosis demonstrated attenuated CVC response to anodal stimulation compared with healthy women (p < 0.01; d = 0.56 at peak CVC). Aspirin abolished the increase in CVC in response to anodal stimulation in both groups (p < 0.01; at peak CVC, d = 2.05 HC, d = 1.44 Endo). There was an interaction effect of treatment and condition such that, due to a greater increase in CVC in the placebo condition in the healthy control group compared with that of the endo, there was a greater effect of aspirin on the healthy control group (p < 0.01). Aggregation characteristics data are represented in Figures 2 - 5 (Total aggregation Fig 2 , Peak aggregation Fig 3 , Rate of aggregation Fig 4 , Time to begin aggregation Fig 5 ). Platelet aggregation characteristics normalized to platelet count are shown in supplementary data figures 1 (placebo) and 2 (aspirin; Williams et al., 2026 ). There was no effect of group on total, peak, rate of, and time to begin aggregation response to Col5, Col1, AA, or Tx (p ≥ 0.10, f ≤ 0.23 all reagents, all outcomes). Total aggregation responses to Col5 (p = 0.18, f = 0.18) and Tx (p = 0.38, f = 0.16) were unaffected by aspirin, but total aggregation response to Col1 (p < 0.0001, f = 0.88) and AA (p < 0.0001, f = 0.96) was reduced following aspirin. Peak aggregation responses to Col5 (p = 0.91, f = 0.18) and Tx (p = 0.53, f = 0.10) were unaffected by aspirin, but peak aggregation responses to Col1 (p < 0.0001, f = 1.09) and AA (p < 0.0001, f = 1.08) were attenuated by aspirin. The rate of aggregation response to Tx was unaffected by aspirin (p = 0.19, f = 0.21), but the rate of aggregation response to Col1 (p < 0.0001, f = 0.74), AA (p < 0.0001, f = .86), and Col5 (p = 0.0002, f = 0.53) were attenuated following aspirin. The time to begin aggregation was delayed in response to all reagents following aspirin (p ≤ 0.01 all reagents; Col5 f = 0.33, Col1 f = 0.48, Tx f = 0.52, AA f = 1.08). Total aggregation response to Col5 (p = 0.03, f = 0.29) and Tx (p = 0.009, f = 0.44) were reduced following TRB incubation, but total aggregation response to Col1 (p = 0.87, f = 0.29) and AA (p = 0.90, f = 0.01) were unaffected by incubation. Peak aggregation response to Col1 (p = 0.59, f = 0.07), AA (p = 0.95, f = 0.00), and Tx (p = 0.34, f = 0.15) were unaffected by incubation, but the peak aggregation response to Col5 was increased following TRB (p = 0.02, f = 0.29). Both the rate of aggregation and time to begin aggregation in response to all reagents was unaffected by TRB (Slope all reagents p ≥ 0.1, f ≤ 0.15; Lag all reagents p ≥ 0.1, f ≤ 0.22). ELISA results are shown in Figure 6 . aspirin attenuated the TxB2 plasma concentration (p = 0.03, f =0.81), but there was no difference in TxB2 concentration between groups (p = 0.27, f = 0.27) and no interaction effect (p = 0.57, f = 0.18). While there was no difference in PGE2 concentration between groups (p = 0.70, f = 0.01) or treatment (p = 0.98, f = 0.24), there was a main interaction effect (p = 0.02, f = 0.58). Calculated plasma volume was not different between groups (4.4 ± 0.34 L HC vs 4.5 ± 0.46 L Endo, p = 0.63). There was no correlation between plasma concentrations of TxB2 (Endo p = 0.06, HC p = 0.14) or PGE2 (Endo p = 0.18, HC p = 0.98) and AUC of the CVC response to anodal stimulation, nor was there a correlation of the AUC of CVC response to AS with platelet count (Endo p = 0.20, HC p = 0.78). Regressions are shown in supplementary figure 3 ( Williams et al., 2026 ).

COX-mediated vasodilation is attenuated in women with endometriosis. Although the precise mechanism underlying this pathophysiology is undetermined, it does not appear to be related to altered platelet activity. While there was no effect of endometriosis on platelet activity, it is noteworthy that TP may play an alternate role in aggregation in whole blood samples than previously considered. Our findings may lend insight into the pathophysiologic mechanism instigating vascular dysfunction in women with endometriosis as well as the basic mechanisms of platelet aggregation in healthy women.

The purpose of the present study was to determine if COX metabolism impacts the mechanisms of vasodilation and platelet aggregation in patients with endometriosis. To investigate this, we utilized a non-specific COX-mediated vasodilator technique, a battery of pro-aggregatory reagents involved in the COX pathway in impedance aggregometry, and quantified the COX metabolites TXB2 and PGE2. Contrary to our hypothesis, women with endometriosis demonstrated attenuated CVC responses to cutaneous anodal stimulation compared with similarly aged healthy women. Despite women with endometriosis having a greater platelet count, there were no differences in TXB2 or PGE2 concentrations, or platelet aggregation characteristics between groups, a finding which persisted after normalizing for platelet count. In both groups, the magnitude of aggregation response to high concentration of collagen was attenuated by inhibition of the thromboxane receptor. The present study demonstrates attenuated current-induced vasodilation in women with endometriosis compared with healthy women. Consistent with previous findings in healthy young adults, the increase in CVC in response to this current application was abolished following aspirin ( Tartas et al., 2005 ; Durand et al., 2002 ) in both groups, confirming this response is entirely mediated by an aspirin-sensitive COX mechanisms, across patients and healthy subjects. The precise mechanism involved in current-induced vasodilation is incompletely understood. It is postulated to occur in some part through an axon reflex involving c-nociceptors and neurogenic inflammation ( Hamdy et al., 2001 ; Tartas et al., 2004; Berliner et al., 1997). If this is the case, our results may indicate a desensitization of the cutaneous afferents via the c-nociceptors in endometriosis. However, given the abundance of data supporting hyperalgesic or allodynic responses in this population ( Aredo et al., 2017 ; Phan et al., 2021 ; Grundström et al., 2018; Fan et al., 2022), we find this an unlikely explanation for the data presented here. Durand and colleagues ( Durand et al., 2002 ) hypothesized a role for platelets in current-induced vasodilation due to the timing of the recovery of the current-induced vasodilation following COX inhibition better aligning with the timeline of COX recovery in the anucleate platelets than that of the nucleated endothelium or vascular smooth muscle cells. However, we did not observe a difference in COX-related platelet function between groups. Due to the timeline of COX inhibition in the present study, we cannot rule out the possibility that the source of the COX metabolites responsible for this vasodilation are related to activity of the vascular endothelium or smooth muscle cells directly ( Hla & Bailey, 1989 ). Our lab and others have shown impaired endothelium-dependent vasodilation in women with endometriosis ( Santoro et al., 2012 ; Dillon et al., 2020) that is further attenuated following oral salsalate intervention (NFκB knockdown) through non-NO-dependent mechanisms ( Williams et al., 2025 ). Collectively, this suggests the NFκB-COX pathway is altered in the vascular endothelium in women with endometriosis; however, the precise interplay between these and the downstream effects remain to be elucidated. In the present investigation, downstream of COX, we show no difference in circulating Tx or PG concentrations between women with endometriosis and healthy women. Despite this, we cannot rule out the possibility that altered expression of COX metabolite receptors on the vascular smooth muscle cells in this population may contribute to impaired COX-mediated vasodilation through alterations in vasoconstrictor tone or impaired vasodilation. It is additionally noteworthy that, although there was not a main effect of condition on PGE2 concentration in plasma, there was a main interaction effect, indicating that the effect of aspirin on total PGE2 is different in endometriosis patients compared with HC ( Figure 6 ). This further suggests alterations in the COX metabolite distribution in endometriosis patients, and further research is needed to determine the precise pathway for attenuated COX-mediated vasodilation. Consistent with the finding of no difference of TBX2 concentrations between groups, we found no differences between groups in the platelet aggregation including total, peak, rate of, and time to begin platelet aggregation, despite differences in platelet counts between groups and previous work identifying more rapid activated partial thromboplastin time without differences in thrombin time ( Wang et al., 2024 ). The lack of difference between groups persisted when normalizing these outcomes for platelet count. In a pre-clinical model of endometriosis, depleting platelets attenuated and perfusing platelets augmented hyperalgesic responses to a noxious thermal stimulus. These data suggest a role for platelet activity and afferent sensitivity in endometriosis ( Ding et al., 2015 ). The evidence for current-induced vasodilation being dependent on platelet activity ( Durand et al., 2002 ), is compelling. However, our findings do not entirely support this as only COX-mediated vasodilation is attenuated in patients with endometriosis, with no differences in platelet aggregation or hyperalgesia ( Williams & Alexander, 2025 ). Moreover, in our thorough investigations into platelet aggregation properties, we found no correlation between the total CVC response (AUC) to AS and platelet count, TxB2, or PGE2 concentrations. Consistent with previous findings, we demonstrate low concentration collagen-induced aggregation is dependent on COX activity ( Williams et al., 2024 ; Carazo et al., 2024 ). We additionally confirm here AA-mediated aggregation is not dependent on TP in a whole blood sample, but is attenuated following COX inhibition. Using this approach we interrogated mechanisms of platelet aggregation within a single testing through these robust pro-aggregatory reagents. Distinctions between the low and high concentration collagen pathways have been discussed elsewhere ( Williams et al., 2024 ), but briefly, if there were a small or moderate effect of the pathophysiology of endometriosis on collagen-induced aggregation, low concentration collagen would likely have been sufficient to reveal it. However, given the inherent variability of impedance aggregometry in a whole blood sample ( Williams et al., 2024 ; Wadowski et al., 2021 ; Femia et al., 2013 ), a more robust aggregatory stimulus – such as with high collagen or arachidonic acid – can be used to interrogate differences in mechanisms of aggregation ( Roberts et al., 2004 ). High concentration collagen was able to induce a greater aggregation (total and peak) once TP were inhibited. This is contradictory to the axiom that the thromboxane pathway in platelets is strictly a positive feedback, pro-aggregatory pathway. The majority of thrombogenesis research utilizes isolated, reductive models to consider the mechanisms of function of independent components of whole blood. Impedance aggregometry, in using whole blood samples, introduces complex interaction in intact tissue. Our finding that TP appear to inhibit aggregation upon exposure to a pro-aggregatory reagent suggests the in-situ role of TP in whole blood limits collagen-induced aggregation. The lack of TP inhibition on the rate or time to begin aggregation in response to high concentration collagen may suggest its moderating role is related to a TP receptor-mediated shape change or prevention of dense granule release that prevents hypercoagulation rather than a downstream enzymatic effect (Rucker et al., 2025). It is noteworthy that while our previous study utilizing a similar approach does not report a significant effect of TRB on high concentration collagen-induced aggregation, an effect of TP inhibition may have been masked by our decision to use a conservative correction for repeated measures and/or the possibility for sex differences, as our previous work was a mixed sample of biological sexes and underpowered to adequately interrogate this variable. The lack of effect of COX inhibition on Tx-induced aggregation confirms the targeted approach of this reagent; the thromboxane mimetic was effective in inducing aggregation downstream of COX enzymatic activity. In either placebo or aspirin, Tx mimetic-induced aggregation was not different between groups. Combined with the finding of no difference in the concentration of circulating TxB2 despite a difference in platelet count, this indicates no difference between women with endometriosis and healthy women in the role of thromboxane in inducing platelet aggregation. Our finding that women with endometriosis – the majority of whom were diagnosed through laparoscopy and are therefore post-surgical excision intervention – demonstrate attenuated non-specific COX-mediated vasodilation provides further support for the need for ongoing multidisciplinary approaches for managing this complex clinical condition. This study provides further support for the negative effect of endometriosis on the cardiovascular system. This finding may have implications in the mechanisms of wound healing in women with endometriosis ( Futagami et al., 2002 ; Eligni et al., 2009 ; Fairweather et al., 2015 ). Finally, increased platelet count and no deviations in platelet aggregation mechanisms confirm that patients with endometriosis are unlikely to require special consideration for clot formation in surgical procedures, although we tested only a limited number of aggregatory agents with a singular, albeit translatable, technique. There are a few limitations to the present investigation to consider. First, although we and others have shown current-induced vasodilation to be entirely COX-mediated, the precise source of this COX (platelet vs. endothelial) modulating this response remains poorly understood. We utilized this technique as a proof-of-concept to interrogate a potential role for COX in the integrative control of the microvasculature. Therefore, further investigation is needed to determine the site of alteration in COX activity that results in attenuated vasodilation. Second, we did not investigate the expression of TP in vascular smooth muscle nor the COX expression in the endothelium. Third, we included participants utilizing a broad range of medications. We cannot rule out the possibility these affected the data; however, inclusion of these participants aids in the ecologic validity of these findings. Future research should utilize targeted approaches to interrogate the downstream COX effectors in neurovascular control. Finally, we did not control for menstrual cycle stage. Women with endometriosis frequently experience irregular menstrual cycles, and treatments for endometriosis (e.g., intrauterine device, contraceptive implant, hysterectomy, etc.) often prevent menses. We therefore tested our participants without regard to menstrual cycle status, allowing a randomization of cycle phase. This approach promotes ecological validity, but is a limitation of this study.

Endometriosis is a female-specific risk factor for cardiovascular disease (CVD). A diagnosis of endometriosis is associated with an increased risk of myocardial infarction and stroke – major adverse cardiovascular events known to be related to atherogenesis and clot formation – by 50% and 39%, respectively ( Farland et al., 2022 ; Mu et al., 2022). Endometriosis is a gynecologic disease estimated to be present in ~10% of women that is characterized by invasive extrauterine endometriotic lesions, difficulty conceiving or carrying a pregnancy, chronic pain, and systemic inflammation ( As-Sanie et al., 2025 ). Women with endometriosis demonstrate endothelial dysfunction, characterized by decreased nitric oxide (NO)-mediated vasodilation, in both the macrovasculature ( Santoro et al., 2012 ; Williams et al., 2025 ) and microvasculature ( Dillon et al., 2022 ; Williams et al., 2025 ). This vascular dysfunction is not improved following broad knockdown of NF-κB-mediated inflammatory pathways ( Williams et al., 2025 ), suggesting alternative or more targeted investigations into the pathophysiology of this vascular dysfunction are required. The activity of cyclooxygenase-2 (COX-2) – a key enzyme in inflammation upregulated by NF-κB activity – is upregulated in endometriosis. Increased expression of COX-2 has been identified in ectopic endometriotic lesions, eutopic endometrium, and peritoneal macrophages in women with endometriosis ( Ota et al., 2001 ; Ding et al., 2015 ; Wu et al., 2002 , Lai et al., 2019 ). Polymorphism for the COX-2 gene has also been identified in women with endometriosis from Asian and South American populations ( Cavalcanti et al., 2016 , Kim et al., 2012 ). While data investigating COX expression and activity in endometriosis within the vasculature specifically is lacking, greater concentrations of IL-1β, IL-6, and TNF-α have been measured in serum samples from women with endometriosis compared with healthy women (Nematian et al., 2017. These cytokines are known to upregulate COX-2 activity through positive feedback ( Ristimaki et al., 1994 ; Dawn et al., 2004 ; Yang et al., 2021 ; Lin et al., 2004 ; Pierce et al., 2009 ). In preclinical models of CVD and in human CVD patients, there is clear evidence for a role for dysregulated COX metabolism – specifically COX-2 – and thromboxane receptors (TP) in sensory afferent sensitization and neurovascular control ( Muller et al., 2015 , Samora et al., 2024 ; Butenas et al., 2021 ; Leal et al., 2011 ; Rollins et al., 2020 ; Smith et al., 2020 ). A downstream metabolite of COX, prostaglandin E2 (PGE2), has been shown to directly sensitize sensory afferents in human dorsal root ganglion neurons ( Davidson et al., 2014 ). Specifically, COX metabolites sensitize sensory afferents to respond at a lower threshold to excitatory stimuli, priming sensory afferents to induce reflex responses more readily. Current-induced vasodilation is a skin-specific technique in which vasodilation is mediated entirely by COX ( Durand et al., 2002 ; Tartas et al., 2004). Current-induced vasodilation in the skin is postulated to occur in some part through an axon reflex involving c-nociceptors and neurogenic inflammation ( Hamdy et al., 2001 ; Tartas et al., 2004; Berliner et al., 1997). C-nociceptors are numerous in the skin ( Hallin et al., 1982 ), and their responsivity to thermal stimuli contribute importantly to cutaneous vasodilation ( Glatte et al., 2019 ). In healthy young adults, topical anesthesia attenuates or prevents iontophoresis-induced increases in cutaneous blood flow, indicating a major role for cutaneous sensory afferents in increased local blood flow induced by this technique ( Berghoff et al., 2002 ). Whether dysregulated COX metabolism affects afferent sensitivity in the cutaneous vascular bed remains to be tested. The COX enzyme is additionally a key mediator of platelet aggregation, an early event in clot formation. Megakaryocytes, the precursor to platelets, express both COX-1 and COX-2 isoforms, with the COX-1 isoform being constitutively expressed throughout the cell lifespan and COX-2 isoform expression being induced in later stages of maturation ( Rocca et al., 2002 ). COX-2 expression in formed platelets is greater in samples taken from patients demonstrating high rates of platelet turnover compared with samples taken from healthy donors. COX-2 expression is upregulated in conditions with greater platelet production ( Rocca et al., 2002 ). Further, COX-2-specific inhibitors attenuated prostanoid production to a greater extent in this population undergoing high platelet turnover than in healthy controls ( Rocca et al., 2002 ). Prostanoids induce a broad array of effects on platelet aggregation; the vasodilators prostacyclin and PGE2 are anti-aggregatory while thromboxane A2 (TxA2) is a feed-forward modulator of platelet recruitment and subsequent clot formation. In ex vivo experiments IL-1β treatment resulted in similar COX-2 expression but greater TxB2 concentration (the stable metabolite of TxA2) in ectopic endometrial stromal cells, although COX-1 metabolism was not investigated in this study ( Ding et al., 2015 ). Women with endometriosis demonstrate a more rapid activated partial thromboplastin time despite no difference in thrombin time ( Wang et al., 2024 ), allowing speculation on the distinct pathophysiological mechanisms of clot formation in this clinical population. Given that women with endometriosis are at increased risk for clot-related adverse cardiovascular events, it is important to understand how platelet function may be impacted by this disease. In a preclinical model of endometriosis, platelet activity is related to hyperalgesia; depleting platelets by injecting GPIbα polyclonal IgG (targeted anti-platelet antibody) improved tolerance to noxious heat over the weeks following endometriosis induction whereas injecting additional platelets worsened tolerance at a more rapid rate than the control group ( Ding et al., 2015 ). This anti-platelet intervention attenuated COX-2 expression ( Ding et al., 2015 ). Women with endometriosis are frequently hyperalgesic or allodynic and have repeatedly demonstrated lowered pressure-pain thresholds ( Grundstrom et al., 2019 ; Phan et al., 2021 ). This heightened sensitivity to noxious stimuli is thought to be related, in part, to COX-mediated neuroinflammation of primary sensory neurons (Fan et al., 2022; Aredo et al., 2017 ). Taken together, these data suggest a possibility for a pathophysiologic role for COX in neurovascular control and mechanisms of platelet function mechanisms in women with endometriosis. Therefore, the purpose of this study was to determine if systemic COX activity in women with endometriosis impacts mechanisms of neurovascular control and platelet aggregation. To examine this, we performed cutaneous anodal current stimulation and impedance aggregometry in women with endometriosis and healthy, similarly aged women in a randomized, blinded, placebo-controlled design using aspirin, a non-selective COX inhibitor. We hypothesized that women with endometriosis would demonstrate augmented blood flow responses to an anodal current stimulus and platelet aggregation compared with healthy women, and that this difference would be mediated, at least in part, by COX activity.

Supplementary material for this manuscript is available at: doi: 10.6084/m9.figshare.31128967